PROJECT SUMMARY Pili are bacterial surface structures that are used by bacteria in many ways. They can be used for motility, genetic exchange, and surface attachment, and are often critical virulence factors for pathogens. A particular type of pilus, the flp pilus, is found in many bacterial pathogens, including Vibrio vulnificus and Aggregatibacter actinomycetemcomitans, and is used by some members of the human microbiome to establish permanence in the intestinal tract. Despite its importance, the flp pilus is not nearly studied to the depth of other pilus systems, and has many unexplored facets and curiosities. In this proposal we outline experiments to explore the flp pilus using a model bacterium that provides many technical benefits for studying pili: Caulobacter crescentus. A common characteristic of the flp system is that the pilin gene is regulated separately from the rest of the pilus genes. In C. crescentus, the transcriptional activator CtrA binds to four sites in the pilin promoter. Preliminary data indicates that one site induces expression, while the remaining sites inhibit and/or delay transcription from their upstream positions, a phenomenon not previously reported in bacteria. Regulation of the pilin promoter will be explored by mutating different CtrA-binding sites and measuring changes in expression timing and magnitude. The direct interaction and potential cooperativity of CtrA binding at the pilin promoter will be assessed using biochemical assays. Lastly, regulation of the pilin subunit will be altered by mutagenesis, and the effect of differential regulation of the pilin subunit and how it contributes to pilus synthesis as well as physiological consequences such as phage infection and surface attachment will be examined (Aim 1). Another common characteristic of the flp pilus is that it is often polarly localized. Localization depends on the TadZ protein, but how that protein is localized is a mystery. Protein interaction techniques will be used to identify and characterize the factors that cause TadZ, and thus the flp pilus, to become polarly localized. Additionally, the entire protein complement of the flp pilus in this organism will be determined by purification of whole intact pili using a novel purification strategy, something that has never before been accomplished (Aim 2). These studies will provide insight into features conserved among flp pili systems, and thus provide insight into a structure important for many human pathogenic and commensal organisms.